Abstract The goal of this project is to generate highly potent trispecific antibodies for the prevention and treatment of botulism due to botulinum neurotoxins (BoNT) serotypes A, B, E, and F. BoNTs are one of the six highest-risk threat agents for bioterrorism, due to their extreme potency and lethality, ease of production, and need for prolonged intensive care. Equine antitoxin has a high incidence of allergic reactions, a short serum half-life leading to reintoxication, is challenging to administer, and cannot be given prophylactically. Given the extraordinary toxicity of BoNTs, antitoxin must be of very high potency. Previous studies have found that no single mAb neutralizes BoNT with the requisite potency, however combining mAbs binding non-overlapping epitopes leads to highly potent BoNT neutralization due to rapid clearance of BoNT from the circulation. We have generated combinations of three mAbs that bind all subtypes of BoNT/A, B, or E and result in highly potent BoNT neutralization in vivo. Based on the potency, HHS has awarded contracts to a biotechnology company (XOMA (US) LLC) to develop these mAb combinations for clinical use. The BoNT/A three mAb combination has entered phase 1 clinical trials. The BoNT/B and BoNT/E three mAb combinations are in cGMP manufacturing and will enter clinical trials by 2013-2014. A challenge of this approach is the large number of mAbs that need to be manufactured and quality controlled (3 per serotype). This adds to the cost and complexity of the product, especially if the mAbs are all going to be combined to create a multi-serotype drug. Our proposed alternative is to create a single trispecific mAb for each serotype incorporating the binding sites of each of the three mAbs. For this work, we hypothesize that such trispecific mAb can be generated which will have the same binding and BoNT neutralization properties as the three mAb combination. In the R21 portion we will construct trispecific antibodies (TsAbs) binding three non-overlapping BoNT/A epitopes. Four different TsAbs will be constructed that differ with respect to where each of the three variable domains are positioned relative to the antibody Fc. Each TsAb will be characterized and optimized with respect to affinity for each of the three epitopes, stoichiometry of binding to BoNT/A, stability, and ease of expression and purification. Multiple TsAb constructs will be evaluated for in vivo pharmacokinetics (PK) and BoNT/A clearance and neutralization potency in the mouse neutralization assay. In vivo studies will be conducted at the USDA by our long-term collaborator, Dr. Luisa Cheng. These studies will identify those constructs with optimal properties and will result in a single mAb that potently neutralizes BoNT/A. In the R33 portion of this grant, the optimal construct identified for BoNT/A will be applied to three other BoNT serotypes, B, E and F. The result of this project will be antibodies that would treat over 99% of human botulism cases.